Adenine, adenosine, adenosine phosphate compounds, DNA, RNA and the like are known as adenyl group-containing substances which take important roles in the living body as composing elements of coenzymes, high-energy phosphate compounds, genes and the like.
In general, measurement of adenine and adenosine by separating them from other nucleic acid bases such as guanine, guanosine and the like is carried out making use of chromatographic separation techniques, generally using high performance liquid chromatography.
On the other hand, measurement of nucleic acids is carried out generally making use-of their ultraviolet absorption in the vicinity of 260 nm. In recent years, an ethidium bromide-aided fluorochrome technique has been developed.
Measurement of a target nucleic acid in samples to be tested is carried out using its complementary nucleic acid which is directly or indirectly labeled with a marker such as a radioactive isotope, an enzyme, a fluorescent material, a chemiluminescent substance or the like.
In a method in which a target nucleic acid in a sample to be tested is detected by a nucleic acid amplification technique making use of a polymerase, detection of the amplified nucleic acid is carried out by employing a combination of a nucleic acid electrophoresis and an ethidium bromide-aided fluorochrome technique.
In addition, specific affinity of an antibody for its corresponding antigen is used in immunological assay methods which are divided into methods in which the immunologicat reaction is directly detected as precipitation or turbidity of the resulting antigen-antibody complex and methods in which the antibody or antigen is labeled with a marker for use in the measurement. In the latter case, a radioactive isotope, an enzyme, a fluorescent material, a chemiluminescent substance and the like are used as markers, and the measurement can be effected with greater sensitivity of detector than in the former case. In recent years, a high sensitivity assay method has been developed in which an antibody is labeled with a nucleic acid and used in combination with a polymerase-aided nucleic acid amplification technique. Also in this method, an ethidium bromide-aided fluorochrome technique is used in its final step for the detection of amplified nucleic acid.
Kuroda et al. have reported at the 41th Annual Meeting of The Japan Society of Analytical Chemistry that quantitative determination of adenine can be attained by a method in which a luminescent substance is derived by allowing phenylglyoxal to react with adenine in the presence of hydrochloric acid, emission from the resulting chemiluminescent substance is effected by adding sodium hydroxide aqueous solution to the reaction solution in the presence of H.sub.2 O.sub.2 and then the resulting luminescence activity is measured. Though this method has disadvantages of causing a high background value in the absence of adenine and its low emission quantity per adenine, it is useful from a viewpoint that it can effect adenine-specific measurement easily without requiring complex separation purification steps which are essential in the aforementioned assay methods.
On the other hand, heteropoly-acids and their salts are used as catalysts, to provide strong acidity and oxidation potential. They are used in a variety of reactions such as oxidation of hydrocarbons, polymerization and epoxidation of alkenes and the like.
The aforementioned assay methods have the following disadvantages.
The method in which adenine and adenosine in a test sample are measured by employing a separation step making use of a chromatography step requires complex handling and additional equipment such as high performance liquid chromatographys. In addition, when an adenine-containing substance is measured, it is necessary to employ a pre-treatment step in order to decompose the adenine-containing substance into adenine or adenosine.
The nucleic acid measuring method which uses ultraviolet absorption in the vicinity of 260 nm has disadvantages in terms of its measuring sensitivity and specificity.
The nucleic acid measuring method by an ethidium bromide-aided fluorochrome technique requires special care in handling, because the fluorescence intensity fluctuates extensively between single-stranded and double-stranded nucleic acids, and ethidium bromide is a strongly carcinogenic substance.
In the nucleic acid measuring method in which a complementary nucleic acid is directly or indirectly labeled, and a target nucleic acid in a sample is hybridized to the complementary nucleic acid, it is necessary to devise a special means with regard to the labeling position and the like when the complementary nucleic acid is labeled so that binding of the target nucleic acid and the complementary nucleic acid is not spoiled.
Even in the cases of the method in which a target nucleic acid in a sample is detected by a polymerase-aided nucleic acid amplification technique and the method in which high sensitivity measurement is effected by labeling an antibody with a nucleic acid by means of an immunoassay technique and then using a nucleic acid amplification technique, it is necessary to use ethidium bromide in their final detection step, hence posing the aforementioned problems.
It is accordingly a primary object of the present invention to resolve one or more of these problems.